H5N8亚型禽流感病毒HA和NA基因双重荧光RT-PCR检测方法的建立  

Establishment of Duplex Fluorescence RT-PCR Method for Detection of H5N8 Subtype Avian Influenza Virus HA and NA Genes

作　　者：马玲 姚贵哲 王准 孟庆峰[1,2] 李玥 肖芳 刘金华 王伟利 MA Ling;YAO Gui-zhe;WANG Zhun;MENG Qing-feng;LI Yue;XIAO Fang;LIU Jin-hua;WANG Wei-li(College of Animal Science and Technology,Jilin Agricultural University,Changchun,Jilin,130118,China;Changchun Customs Technology Center,Changchun,Jilin,130062,China)

机构地区：[1]吉林农业大学动物科学技术学院,吉林长春130118 [2]长春海关技术中心,吉林长春130062

出　　处：《动物医学进展》2020年第6期32-37,共6页Progress In Veterinary Medicine

基　　金：吉林省重点科技攻关项目(20150204029NY)。

摘　　要：H5N8亚型禽流感是传播性极强的人兽共患病,为了简便快速的检测H5N8亚型禽流感病毒,针对H5N8亚型禽流感病毒HA基因和NA基因序列分别设计合成特异性引物和探针,通过优化反应条件,建立了同一反应程序下同时扩增HA基因和NA基因的双重荧光RT-PCR检测方法。结果显示,该检测方法特异性强,不与其他亚型禽流感病毒和禽常见病原发生交叉反应,敏感性可达10 copies/μL;组内组间重复性好,平均变异系数均小于0.4%;用该方法对108份临床样品检测,结果与病毒分离鉴定方法检出率一致。综上所述,建立的H5N8亚型禽流感病毒双重荧光RT-PCR检测方法具有快速、特异、敏感、重复性好等优点,为H5N8亚型禽流感的快速诊断和疫情防控提供了一定的技术支持。H5N8 subtype avian influenza virus is a highly transmitted infectious disease and zoonotic disease.In order to diagnose the H5N8 subtype avian influenza virus easily and quickly,this study designed and synthesized specific primers and probes for the H5N8 subtype avian influenza virus HA gene and NA gene sequences respectively.By optimizing the reaction conditions,a duplex fluorescence RT-PCR method for simultaneous amplification of HA gene and NA gene under the same reaction procedure was established.The results showed that the method was specific,did not cross-react with other subtypes of avian influenza virus and common pathogens in poultry,and had a sensitivity of up to 10 copies/μL;the intragroup and intergroup repeatability tests were good,and the average variation coefficient was less than 0.4%.108 clinical samples were detected by this method,the detection rate of virus isolation and this method was consistent.In summary,this study established the H5N8 subtype avian influenza virus duplex fluorescence RT-PCR detection method,with the rapid,specific,sensitive advantages,and good repeatability,for the rapid diagnosis of H5N8 subtype avian influenza and the prevention and control of the disease.

关 键 词：H5N8亚型 禽流感病毒 HA基因 NA基因 荧光RT-PCR 

分 类 号：S852.659.5[农业科学—基础兽医学]